Application of pcsk9 inhibitors in the preparation of drugs for the treatment of inflammatory and immune diseases

ABSTRACT

The invention belongs to the biopharmaceutical. It involves the role and mechanism of PCSK9 in inflammatory immune diseases, and the application of PCSK9 inhibitors to the preparation of drugs for the treatment of inflammatory immune diseases mediated by T cells. In particular, it involves the use of PCSK9 monoclonal antibody, PCSK9 interference RNA and PCSK9 small molecule inhibitors for treating psoriasis, atopic dermatitis, or urticaria. The invention uses psoriasis as an embodiment for the study of inflammatory and immune diseases. It is found that PCSK9 plays an important role in the treatment of inflammatory immune diseases. The PCSK9 monoclonal antibody, PCSK9 interference RNA, and PCSK9 small molecule inhibitor can be further developed for treating inflammatory immune-diseases, such as psoriasis with fewer adverse reactions, at low cost, and with good efficacy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/965,907, filed Apr. 28, 2018, pending, which is acontinuation-in-part of International Pat. Appl. No. PCT/2016/111613,filed on Dec. 23, 2016, which claims priority to Chinese Pat. Appl. No.201510728438.6, filed on Oct. 29, 2015.

I. THE TECHNICAL FIELD

The invention belongs to the field of biopharmaceutical technology,including the role and mechanism of PCSK9 in inflammatory immunediseases and the application of PCSK9 inhibitors in the preparation ofdrugs for treating inflammatory immune diseases.

II. BACKGROUND

The incidence of inflammatory immune diseases is high, and there are atleast hundreds of millions of people worldwide, including manyautoimmune diseases, such as psoriasis, eczema (atopic dermatitis),lupus erythematosus, rheumatoid arthritis, dermatomyositis, scleroderma,Crohn's Disease, etc.). Because these diseases can involve multipleorgans, leading to organ injury of heart, liver, kidney, blood vessel,lung, joint and brain. Its mortality rate is second only to malignanttumor. In view of the fact that the etiology and pathogenesis of thesediseases is quite complex and cannot be cured at present.Glucocorticoids and immunosuppressants are the main therapeutic drugscommonly used in clinical practice. The effective rate of these drugs isonly about 50%. And the severe adverse side effect, which include bonemarrow suppression, liver and kidney dysfunction, osteoporosis,susceptibility to infection and tumors, greatly limit long-term use ofsuch drugs. In recent years, the biologics have gradually become thepopular direction of drug research and development due to theselectivity of the therapeutic targets and the little side effects. Butso far, only a few of these biologic drugs have achieved great efficacy.At the same time, high prices limit wide application.

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of theproprotein convertase family, which is secreted as an inactive proenzymein the liver. The cDNA of the PCSK9 gene is 3617 bp, which encodes aPCSK9 protein with 692 amino acids. The PCSK9 precursor separatedN-terminal predomain by intramolecular catalyzing, then the separatedN-terminal anterior region is connected to catalytic region to allowmature PCSK9 proteins to leave the endoplasmic reticulum and enter thesecretory pathway. After PCSK9 is secreted into the extracellular space,it binds at the cell surface to the low density lipoprotein (LDL)receptor in the first epidermal growth factor like domain. Followingbinding, PCSK9-LDL binding receptor complex enters lysosome to degrade,which leads to the decrease of LDL receptors on the cell surface.Therefore, the level of PCSK9 is negatively correlated with the level ofLDL receptor. Several studies have shown that the mutation of PCSK9 genecan significantly decrease the LDL cholesterol level and the incidenceof coronary heart disease in different patient groups.

In view of the significant effect of inhibition of PCSK9 on reducing LDLcholesterol and coronary disease, a number of treatment schemes havebeen developed to deactivate PCSK9, for the purpose of reducing LDLcholesterol and coronary disease. Because of its high efficiency, targetselectivity, and good stability, monoclonal antibodies have become a hotspot in new drug research. Two PCSK9 monoclonal antibodies have recentlybeen approved by FDA and the European Medicines Agency (EMA) fortreating hypercholesterolemia in patients who are still unable to reduceLDL by current treatments.

The PCSK9 monoclonal antibodies that have been launched to treathypercholesterolemia include Alirocumab (SAR236553/REGN727, Sanofi &Regeneron Pharma) and Evolocumab (AMG 145, Amgen). Preclinical trials ofother PCSK9 monoclonal antibodies, such as Pfizer's Bococizumab (asRN316/PF 04950615) and LY3015014 (Eli Lilly and Company), have beencompleted. Overall, the clinical research found that the patients withhypercholesterolemia treated with these drugs had good tolerance, andthere was no significant difference in the incidence of adversereactions between the placebo group and the active treatment group.

The main target for inhibition of PCSK9 secretion is mRNAs, which can beobtained by using antisense oligonucleotides (ASOS) to obtain theirshort sequences, which can be given intravenously in the form of smalllipid nanoparticles. In rats, liver-specific siRNA targeting PCSK9 couldmake the maximum silencing efficiency at 50-60%. The plasma LDLcholesterol level decreased by 30%. In non-human primates, a dose of 5mg drug reduced LDL cholesterol by 56-70% after 72 hours and lasted for3 weeks. In phase I trial, the siRNA oligonucleotide ALN-PCS02 wasinvestigated. Compared with placebo, the drug can quickly reduce thelevel of PCSK9 (average 68%) and LDL cholesterol (average 41%) in adose-dependent manner. The drug is safe and has no obvious adverseeffects.

For PCSK9 small molecule inhibitors, Pfizer is developing a PCSK9 smallmolecule inhibitor. In animal experiments, the drug can significantlyreduce LDL cholesterol level. In addition, the company is designing avaccine drug that patients need only once a year to achieve a long-termreduction in LDL.

However, so far, all new research and drug development projects relatedto PCSK9 inhibitors have been based on their role in reducing LDLcholesterol level and coronary disease. So far, there has been noreporting on the use of PCSK9 inhibitors in the treatment ofinflammatory and immune diseases.

It is well known that the incidence of inflammatory immune diseases ishigh; their pathogenesis is complex and they include many diseases.However, there are often similar etiological or pathological bases amongdifferent inflammatory immune diseases. They are usually treated withthe same drugs: glucocorticoids and immunosuppressants, which are usedmost frequently in clinical practice. For the development of new drugs,the high degree of similarity in the treatment and medication of varioustypes of inflammatory immune diseases. so a disease model that is easyto observe and analyze the outcome of the treatment is usually chosen tocarry out the preliminary study. At present, because the therapeuticeffect of psoriatic lesions is easy to observe, it has become theexperimental field to generally study the treatment of inflammatoryimmune diseases. Psoriasis is an immunological disease mediated by Tcells with multiple genetic mutation background. It is susceptible to beassociated with metabolic syndrome (hypertension, hyperlipidemia,hyperglycemia and obesity) and cardiovascular disease. The disease isprone to relapse and requires lifelong treatment. Psoriasis mainlyincludes four types: psoriasis vulgaris, psoriasis pustulose, psoriasiserythematosus, and psoriasis arthritis. According to the severity it isdivided into mild, moderate, severe. At present, immunosuppressants andretinoic acid are commonly used in the treatment of severe psoriasis.Adverse reactions are common, including bone marrow suppression, liverand kidney injury and hyperlipidemia. Psoriatic Arthritis (PsA) is thesecond most common inflammatory joint disease, which can lead todisability. Compared with rheumatoid arthritis (RA), PsA lacksappropriate treatment drugs. Antirheumatic drugs for improving thecondition of the disease such as Methotrexate, leflunomide and otherantirheumatics, which lack randomized experimental evidence, have beenthe first line PsA treatment. With the research progress on thepathogenesis of the disease, biological agents, which are the keytargets of the pathogenesis of the disease have been gradually used inclinical treatment. At present, biological agents used in the treatmentof psoriasis and PsA mainly include monoclonal antibodies (mAbs) againstTNF-α, a target of immune pathogenesis pathway. They include infleximmonoclonal antibody (trade name: Remicade), adamumab injection (tradename: Merlot), human recombinant II tumor necrosis factor receptorantibody fusion protein for injection (trade name: Etanercept), andUlinumab, which targets IL-12 and IL-23 common subunit P40. However,about 30% of patients may show poor or ineffective response to thesedrugs, and long-term use also carries the risk of inducing infection(including tuberculosis) and tumors. Both the European Drug Agency (EMA)and the Food and Drug Administration (FDA) found that there seemed to beno better treatment for those who don't respond to the currentlyavailable drug. In conclusion, none of the currently available drugs caneffectively improve metabolic disorders in psoriatic patients. So far,no therapeutic target has been found to treat psoriatic lesions andmetabolic abnormalities.

III. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Mild skin inflammation was observed in IMQ treatment region ofPCSK9 knockout mice. After 5 days of continuous coating, markederythema, thickening of scales and lesions were observed and measured inthe coating area of at the back of C57BL/6 mice. In PCSK9 knockout mice,there were only slight erythema, thickening of scales and lesions.

FIG. 2: The score of skin inflammation (erythema+scale+infiltration) inPCSK9 knockout mice were significantly lower than that in control group(P<0.05). A daily score was given on the skin lesions of the drug-coatedarea on the back of the mice, the erythema, scale, lesion thickening andtotal score of C57BL/6 mice were significantly higher than those ofPCSK9 gene knockout mice (P<0.05).

FIG. 3: The histopathological changes of skin in the IMQ treatmentregion of PCSK9 knockout mice were closer to normal. After 5 days'coating, the skin tissue (treated and non-treated) of mice back werestained with HE. The skin of IMQ treatment area of C57BL/6 mice showedtypical psoriasis pathological changes, such as skin thickening,dermatoid lengthening, thickening of spinous layer, hyperkeratosis withincomplete keratosis, Kogoj abscess and Munro abscess. However, the skinof PCSK9 gene knockout mice showed mild epidermis thickening andhyperkeratosis, no extension of dermatoid, thickening of spinous layer,hyperkeratosis with incomplete keratosis, and typical psoriaticpathological changes, such as Kogoj abscess and Munro abscess. Theuntreated skin of both groups showed normal skin structure.

FIG. 4: Immunofluorescence assay showed: Compared with PCSK9 knockoutmice, the expression of PCSK9 in the control group was upregulated inthe skin of the inflammatory area. The expression of PCSK9 in IMQtreated area of C57BL/6 mice was significantly higher than that inuntreated area. However, there was no PCSK9 expression in the skin ofIMQ treated and untreated regions of PCSK9 knockout mice.

FIG. 5: The expression of NF-kB in the skin of PCSK9 knockout mice wassignificantly lower than that of the control group. The expression ofNF-kB in the skin of C57BL/6 mice and PCSK9 knockout mice wassignificantly higher than that of the untreated regions in the skin ofC57BL/6 mice and PCSK9 knockout mice respectively. However, theexpression of NF-kB in the skin of PCSK9 knockout mice was significantlylower than that of C57BL/6 mice.

FIG. 6: si-PCSK9 transfection significantly inhibited cell viability ofhuman keratinocytes. The survival number of primary keratinocytestransfected with si-PCSK9 was significantly lower than that of si-Contransfection (P<0.001).

FIG. 7: si-PCSK9 transfection significantly promoted apoptosis andinhibited the proliferation of human keratinocytes. The apoptosis ofprimary keratinocytes transfected with si-PCSK9 increased significantly(P<0.05), while proliferating (the proportion of S+G2/M phase cells)decreased significantly (P<0.05).

FIG. 8: Immunohistochemically staining showed that the expression ofPCSK9 in psoriatic lesions was significantly higher than that inpsoriatic non-lesions and normal controls. PCSK9 positive expressioncells were mainly distributed in epidermis, and dermis near theepidermis, but not in dermis vessels.

FIG. 9: The results of Q-PCR (quantative PCR) showed that the expressionof PCSK9 in psoriatic lesions (PP) group was significantly higher thanthat in psoriatic non-lesion (PN) group and normal control (NN) group(P<0.001).

FIG. 10: The expression of PCSK9 in peripheral blood of patients withpsoriasis, eczema and urticaria was significantly higher than that ofnormal controls (P<0.05).

FIG. 11: PCSK9 protein can significantly promote the secretion of IL-17,IL-2 and IFN-Gamma (P<0.01) by CD4 T cells isolated from peripheralblood of patients with psoriasis, eczema and urticaria.

FIG. 12: Subcutaneous injection of PCSK9 small molecule inhibitor andPCSK9 monoclonal antibody can reduce the score of erythema, scale andinfiltration induced by IMQ in mice skin (P<0.01).

FIG. 13: External use of PCSK9 small molecule inhibitors and PCSK9monoclonal antibodies can significantly reduce the score of skinerythema, scale, and infiltration of IMQ induced mice. The efficacy ofPCSK9 small molecule inhibitor is better than that of PCSK9 monoclonalantibody (P<0.01).

FIG. 14: Compared with control siRNA(siCon), siPCSK9 could significantlyreduce the expression of PCSK9 in mouse skin one day after theapplication, and the inhibition rate was more than 80%.

FIG. 15: siPCSK9 can inhibit the expression of NF kappa B, IL-17, IL-22,IL-23 and other inflammatory factors after a day of skin treatment.

FIG. 16: After induction of IMQ, the skin psoriasis lesion, erythema,scale and infiltration score of mice treated with siPCSK9 weresignificantly lower than those of the control group (P<0.01).

FIG. 17: On days 2-6 after induction of IMQ, compared with the controlgroup, the skin erythema, scales and infiltration of BALB mice inducedby IMQ were significantly inhibited by siPCSK9.

IV. DETAILED DESCRIPTION OF THE INVENTION Technical Issues

The problems to be solved in the present invention: the role andmechanism of PCSK9 in the treatment of inflammatory immune diseases andthe application of PCSK9 inhibitors in the preparation of drugs for thetreatment of inflammatory immune diseases. The invention uses psoriasisas an example for the study of inflammatory immune diseases, and findsthat PCSK9 plays an important role in the treatment of a variety ofinflammatory immune diseases.

Technical Solution

We first found that PCSK9 plays an important role in the treatment of avariety of inflammatory immune diseases. We used PCSK9 knockouttransgenic mice to establish psoriatic like inflammatory model inducedby imiquimod, which proves that inhibition of PCSK9 has a very obvioustherapeutic effect on psoriatic inflammatory lesions. A study on themechanism of inhibiting PCSK9 in the treatment of inflammatory immunediseases by cultured human keratinocytes found that knock-down of PCSK9expression by siRNA could significantly inhibit the abnormalproliferation of keratinocyte and promote its apoptosis through NFkbpathway.

To further confirm the role of PCSK9 in the pathogenesis of psoriasis,the invention collected lesions and non-lesions from 30 psoriasispatients, and skin samples from 30 normal human subjects. Theimmunohistochemistry results showed: the expression of PCSK9 inpsoriatic lesions was significantly higher than that in non-lesions andnormal controls (p<0.05). PCSK9 positive cells were mainly distributedin epidermis and dermis near the epidermis, but not in dermis vessels.Detected by real-time fluorescence quantitative nucleic acidamplification detection system (Q-PCR), it was found that the expressionof PCSK9 was found in the skin of both patients and normal subjects, butit was significantly higher in the psoriasis patients than in the normalcontrols (P<0.01). No expression of PCSK9 was found in the peripheralblood mononuclear cells (PBMC) of patients with psoriasis, eczema, andurticaria and the control group by Q-PCR. However, the expression ofPCSK9 in blood in patients with psoriasis, eczema and urticaria wassignificantly higher than that in the control group by ELISA (P<0.05).

Next, we isolated CD4 T cells from peripheral blood of patients withactive psoriasis, eczema and urticaria and found PCSK9 protein couldsignificantly promote the secretion of IL-17, IL-2 and IFN-Gamma by CD4T cells, and increased the expression of NFkb, especially in psoriaticpatients.

In all PCSK9 inhibitors, representative monoclonal antibodies, smallinterfering RNA and small molecular inhibitors were selected herein.Treated psoriasis-like mice model by subcutaneous injection (the dose isthe same as the known LDL cholesterol experiment) in comparison withcontrol group. The results showed that the therapeutic effect of PCSK9monoclonal antibody group and PCSK9 small molecule inhibitor group wassignificantly better than that of control group. There were no obviousadverse reactions in PCSK9 monoclonal antibody group and PCSK9 smallmolecule inhibitor group. The results showed that the systemic use ofPCSK9 small molecule inhibitors and PCSK9 monoclonal antibodies hadobvious therapeutic effects on psoriasis-like inflammation and thetherapeutic effect was significantly better than that of the controlgroup.

In some embodiments, representative PCSK9 small molecule inhibitors andPCSK9 monoclonal antibodies were prepared for use as an externalointment (drug concentration 0.001-0.05%) and applied to IMQ inducedinflammatory psoriasis like lesions in mice once a day in comparisonwith external ointment without drugs as controls. The results showedthat the efficacy of PCSK9 monoclonal antibody group and PCSK9 smallmolecule inhibitor group was significantly better than that of controlgroup. The effect of PCSK9 small molecule inhibitor group was betterthan that of PCSK9 monoclonal antibody group, and there was no obviousadverse reaction in each group. It was proved that topical use of PCSK9small molecule inhibitor and PCSK9 monoclonal antibody had obvioustherapeutic effect on psoriasis-like inflammation and their efficacy wasbetter than that of control group.

In some embodiments, representative PCSK9 small molecule interferenceRNA (siPCSK9) was applied to IMQ induced psoriasis like inflammatorylesions in mice, once a day. Compared with the control group, thelesions of the siPCSK9 group were improved obviously, and no obviousadverse reaction was found.

For the first time, it was found that: 1. Both systemic and topical useof PCSK9 monoclonal antibody, PCSK9 small molecule inhibitor, andsiPCSK9 have therapeutic effects on psoriatic lesions, which aresignificantly superior to those of the control group. In someembodiments, the invention relates to the treatment of psoriatic lesionby a PCSK9 inhibitor selected from the group consisting ofrepresentative PCSK9 monoclonal antibody, PCSK9 small molecule inhibitorand siPCSK9.

Because of its high inhibiting efficiency, selectivity and goodefficacy, monoclonal antibody has been widely used in the research ofnew drugs. Our study also found that, small molecular inhibitors andsmall interfering RNA aimed at PCSK9 were more effective than monoclonalantibodies in the treatment of psoriasis-like inflammation. Generally,monoclonal antibodies have fewer side effects than small molecularinhibitors, and are more stable than small interfering RNA preparations.However, according to our data, small molecular PCSK9 inhibitors andsiPCSK9 not only have a good efficacy in the treatment of psoriasis likeinflammation, but also have mild side effects.

It is well known to one skilled in this field that, based on the abovemechanism of PCSK9, PCSK9 inhibitors have therapeutic effect on other Tcell mediated chronic immune diseases. These include, but are notlimited to, psoriasis, psoriatic arthritis, eczema (atopic dermatitis),urticaria, glucocorticoid dependent dermatitis, rheumatoid arthritis,scleroderma, diabetes, chronic liver disease and lymphoma, etc. In someembodiments, the invention relates to the treatment of a T-cellmedicated chronic immune disease, wherein the T-cell medicated chronicimmune disease is selected from psoriasis, psoriatic arthritis, eczema(atopic dermatitis), urticaria, glucocorticoid dependent dermatitis,rheumatoid arthritis, scleroderma, diabetes, chronic liver disease, andlymphoma; wherein the PCSK9 inhibitor is a PCSK9 small moleculeinhibitor, a PCSK9 monoclonal antibody or a siPCSK9; wherein the PCSK9inhibitor can be used alone or in combination with other therapeuticagents, including traditional drugs and other new biological agents.

In some embodiments, PCSK9 inhibitor comprising the PCSK9 monoclonalantibody, PCSK9 small molecule interfered RNA and PCSK9 small moleculeinhibitors are used for treating psoriasis, atopic dermatitis orurticaria. In some embodiment, the PCSK9 inhibitor was applied topicallyon the skin.

In an embodiment, the invention relates to a method of treatingpsoriasis by topical use of a PCSK9 inhibitor, wherein the PCSK9inhibitor is selected from the group consisting of a PCSK9 monoclonalantibody, a siPCSK9 and a PCSK9 small molecule inhibitor. In anembodiment, the PCSK9 inhibitor is a PCSK9 vaccine.

In an embodiment, the inhibition of PCSK9 expression by siPCSK9 cansignificantly inhibit the abnormal proliferation of human keratinocytesand promote their apoptosis through NFkb pathway. In an embodiment, theinvention relates to a method of treating keratinocytes by the use ofsiPCSK9.

In an embodiment, the PCSK9 small molecule inhibitor is selected fromthe group consisting of

In an embodiment, the PCSK9 small molecule inhibitor is selected fromthe compounds listed in Table 1. The patent application number cited inTable 1 are incorporated by reference in their entirety.

TABLE 1 Compound No. Structural formula CAS patent application No. 1

1632250- 49-7 WO2014170786 CN105143203 2

1629166- 02-4 US20150376139 WO2016107603 WO2016107602 WO2014150395 3

SBC115076 US2014022957 CN105228616 4

1962177- 03-2 WO2016107603 5

1000339- 97-8 WO2017034997 6

7

2087490- 99-9 WO2017034994 8

2087440- 16-0 WO2017034990 WO2017034994 9

423148- 46-3 WO2017222953 10

130305- 64-5 EP372776

In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody selected fromthe group consisting of Alirocumab, Evolocumab, Bococizumab, DS-001,CA-001, MS-001, LY3015014, NS-001, and antigen binding fragments,variants, conjugates or biosimilars thereof.

In an embodiment, the PCSK9 inhibitor is Alirocumab or antigen bindingfragments, variants, conjugates or biosimilars thereof. In anembodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ IDNO:1 and SEQ ID NO:2. In an embodiment, the PCSK9 inhibitor is a PCSK9antibody comprising sequences at least 99% identical to SEQ ID NO:1 andSEQ ID NO:2. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 95% identical to SEQ ID NO:1 and SEQ IDNO:2. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 90% identical to SEQ ID NO:1 and SEQ IDNO:2.

In an embodiment, the PCSK9 inhibitor is Evolocumab or antigen bindingfragments, variants, conjugates or biosimilars thereof. In anembodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In an embodiment, thePCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99%identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Inan embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprisingsequences at least 95% identical to SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5 and SEQ ID NO:6. In an embodiment, the PCSK9 inhibitor is a PCSK9antibody comprising sequences at least 90% identical to SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

In an embodiment, the PCSK9 inhibitor is Bococizumab or antigen bindingfragments, variants, conjugates or biosimilars thereof. In anembodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment,the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least99% identical to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ IDNO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 95% identical to SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is aPCSK9 antibody comprising sequences at least 90% identical to SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

In an embodiment, the PCSK9 inhibitor is DS-001 or antigen bindingfragments, variants, conjugates or biosimilars thereof. In anembodiment, DS-001 is a PCSK9 antibody comprising SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, and SEQ ID NO:19. In an embodiment, the PCSK9inhibitor is a PCSK9 antibody comprising sequences at least 99%identical to SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprisingsequences at least 95% identical to SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, and SEQ ID NO:19. In an embodiment, the PCSK9 inhibitor is aPCSK9 antibody comprising sequences at least 90% identical to SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

In an embodiment, the PCSK9 inhibitor is CA-001 or antigen bindingfragments, variants, conjugates or biosimilars thereof. In anembodiment, CA-001 is a PCSK9 antibody comprising SEQ ID NO:20, and SEQID NO:21. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 99% identical to SEQ ID NO:20, and SEQ IDNO:21. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 95% identical to SEQ ID NO:20, and SEQ IDNO:21. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 90% identical to SEQ ID NO:20, and SEQ IDNO:21.

In an embodiment, the PCSK9 inhibitor is MS-001 or antigen bindingfragments, variants, conjugates or biosimilars thereof. In anembodiment, MS-001 is a PCSK9 antibody comprising SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 99% identical to SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 95% identical to SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 90% identical to SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQID NO:33.

In an embodiment, the PCSK9 inhibitor is LY3015014 or antigen bindingfragments, variants, conjugates or biosimilars thereof. In anembodiment, LY3015014 is a PCSK9 antibody comprising SEQ ID NO:34 andSEQ ID NO:35. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 99% identical to SEQ ID NO:34 and SEQ IDNO:35. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 95% identical to SEQ ID NO:34 and SEQ IDNO:35. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibodycomprising sequences at least 90% identical to SEQ ID NO:34 and SEQ IDNO:35.

In an embodiment, the PCSK9 inhibitor is NS-001 or antigen bindingfragments, variants, conjugates or biosimilars thereof. In anembodiment, NS-001 is a PCSK9 antibody comprising SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41. In anembodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequencesat least 99% identical to SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQID NO:39, SEQ ID NO:40, and SEQ ID NO:41. In an embodiment, the PCSK9inhibitor is a PCSK9 antibody comprising sequences at least 95%identical to SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQID NO:40, and SEQ ID NO:41. In an embodiment, the PCSK9 inhibitor is aPCSK9 antibody comprising sequences at least 90% identical to SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQID NO:41.

TABLE 2 SEQ ID NO. Description Sequence 1 AlirocumabGlu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyVariable zone inGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser heavy chainSer His Trp Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Val Ala Asn Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr ValAsp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala LysAsn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys Ala Arg Asp Ile Val Leu Met Val Tyr AspMet Asp Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly ThrThr Val Thr Val Ser Ser 2 AlirocumabAsp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro GlyVariable zone inGln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Serlight chain Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly GlnSer Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly ValPro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuLys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys MetGln Thr Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys3 Evolocumab Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro GlyVariable zone inGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser heavy chainSer Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Ser Tyr AlaAsp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala LysAsn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Phe Cys Ala Arg Asp Tyr Asp Phe Trp Ser Ala TyrTyr Asp Ala Phe Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 4EvolocumabGln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly GlnVariable zone inArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Glylight chain Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro LysLeu Leu Ile Ser Gly Asn Ser Asn Arg Pro Ser Gly Val Pro AspArg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile ThrGly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser TyrAsp Ser Ser Leu Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu5 Evolocumab Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro GlyVariable zone inAla Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Serheavy chainTyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gly Gly Leu Glu TrpMet Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr SerThr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr AlaVal Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Trp Gly GlnGly Thr Thr Val Thr Val Ser Ser 6 EvolocumabGln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly GlnVariable zone inSer Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyrlight chain Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro LysLeu Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn ArgPhe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser GlyLeu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Tyr ThrSer Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 7Bococizumab Ser Tyr Tyr Met His Heavy chain: CDR1 8 BococizumabGly Tyr Thr Phe Thr Ser Tyr Tyr Met His Heavy chain: CDR1 9 BococizumabGly Tyr Thr Phe Thr Ser Tyr Heavy chain: CDR1 10 BococizumabSer Pro Phe Gly Gly Arg Heavy chain: CDR2 11 BococizumabGlu Ile Ser Pro Phe Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Heavy chain:Lys CDR2 12 Bococizumab Glu Arg Pro Leu Tyr Ala Ser Asp Leu Heavy chain:CDR3 13 Bococizumab Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu AlaLight chain: CDR1 14 Bococizumab Ser Ala Ser Tyr Arg Tyr ThrLight chain: CDR2 15 Bococizumab Gln Gln Arg Tyr Ser Leu Trp Arg ThrLight chain: CDR3 16 DS-001Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr LeuLys Ala Met Thr Val Asp Arg Phe Lys Ile Ala Ser Trp Pro Arg SerVal Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu GluAla Lys Val Thr Met Asn Trp Trp Gly Arg Ser Gln Gln Val LysAla Val Leu Glu Arg Thr Asp Glu Pro Gly Lys Tyr Thr Ala GlnGly Asp Arg His Val Ala Tyr Ile Ile Arg Ser Pro Val Lys Asp HisTyr Ile Phe Tyr Ser Glu Gly Asn Leu Gln Gly Glu Thr Val ProGly Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn Leu Glu AlaLeu Glu Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu Ser ThrGlu Ser Ile Leu Ile Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly 17 DS-001Gly Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp TyrLeu Lys Ala Met Thr Val Asp Arg Phe Lys Ile Ala Ser Trp ProArg Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly AsnLeu Glu Ala Lys Val Thr Met Asn Trp Trp Gly Arg Ser Gln GluVal Lys Ala Val Leu Glu Arg Thr Asp Glu Pro Gly Lys Tyr ThrAla Gln Gly Asp Arg His Val Ala Tyr Ile Ile Arg Ser His Val LysAsp His Tyr Ile Phe Tyr Ser Glu Gly Asn Leu Gln Gly Glu ThrVal Pro Gly Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn LeuGlu Ala Leu Glu Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly LeuSer Thr Glu Ser Ile Leu Ile Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly 18DS-001 Ala Ser Asp Glu Glu Ile Glu Asp Val Ser Gly Thr Trp Tyr LeuLys Ala Met Thr Val Asp Arg Phe Lys Ile Ala Ser Trp Pro Arg SerVal Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu GluAla Lys Val Thr Met Asn Trp Trp Gly Arg Ser Gln Gln Val LysAla Val Leu Glu Arg Thr Asp Glu Pro Gly Lys Tyr Thr Ala GlnGly Asp Arg His Val Ala Tyr Ile Ile Arg Ser Pro Val Lys Asp HisTyr Ile Phe Tyr Ser Glu Gly Asn Leu Gln Gly Glu Thr Val ProGln Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn Leu Glu AlaLeu Glu Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu Ser ThrGlu Ser Ile Leu Ile Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly GlyGly Gly Ser Leu Ala Glu Ala Lys Glu Ala Ala Asn Ala Glu LeuAsp Ser Tyr Gly Val Ser Asp Phe Tyr Lys Arg Leu Ile Asp LysAla Lys Thr Val Glu Gly Val Glu Ala Leu Lys Asp Ala Ile LeuAla Ala Leu Pro Gly 19 DS-001Gly Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp TyrLeu Lys Ala Met Thr Val Asp Arg Phe Lys Ile Ala Ser Trp ProArg Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly AsnLeu Glu Ala Lys Val Thr Met Asn Trp Trp Gly Arg Ser Gln GluVal Lys Ala Val Leu Glu Arg Thr Asp Glu Pro Gly Lys Tyr ThrAla Gln Gly Asp Arg His Val Ala Tyr Ile Ile Arg Ser His Val LysAsp His Tyr Ile Phe Tyr Ser Glu Gly Asn Leu Gln Gly Glu ThrVal Pro Gly Val Trp Leu Val Gly Arg Asp Pro Lys Asn Asn LeuGlu Ala Leu Glu Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly LeuSer Thr Glu Ser Ile Leu Ile Pro Arg Gln Ser Glu Thr Ser ProGly Lys Leu Gly Gly Gly Gly Ser Leu Ala Glu Ala Lys Glu AlaAla Asn Ala Glu Leu Asp Ser Tyr Gly Val Ser Asp Phe Tyr LysArg Leu Ile Asp Lys Ala Lys Thr Val Glu Gly Val Glu Ala LeuLys Asp Ala Ile Leu Ala Ala Leu Pro Gly Lys Leu Asn 20 CA-001Met Gly Arg Gln Leu Ala Gly Cys Gly Asp Ala Gly Lys Lys AlaSer Phe Lys Met Ser Thr Val His Glu Ile Leu Cys Lys Leu SerLeu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val LysAla Tyr Thr Asn Phe Asp Ala Glu Arg Asp Ala Leu Asn Ile GluThr Ala Ile Lys Thr Lys Gly Val Asp Glu Val Thr Ile Val Asn IleLeu Thr Asn Arg Ser Asn Ala Gln Arg Gln Asp Ile Ala Phe AlaTyr Gln Arg Arg Thr Lys Lys Glu Leu Ala Ser Ala Leu Lys SerAla Leu Ser Gly His Leu Glu Thr Val Ile Leu Gly Leu Leu LysThr Pro Ala Gln Tyr Asp Ala Ser Glu Leu Lys Ala Ser Met LysGly Leu Gly Thr Asp Glu Asp Ser Leu Ile Glu Ile Ile Cys Ser ArgThr Asn Gln Glu Leu Gln Glu Ile Asn Arg Val Tyr Lys Glu MetTyr Lys Thr Asp Leu Glu Lys Asp Ile Ile Ser Asp Thr Ser Gly AspPhe Arg Lys Leu Met Val Ala Leu Ala Lys Gly Arg Arg Ala GluAsp Gly Ser Val Ile Asp Tyr Glu Leu Ile Asp Gln Asp Ala ArgAsp Leu Tyr Asp Ala Gly Val Lys Arg Lys Gly Thr Asp Val ProLys Trp Ile Ser Ile Met Thr Glu Arg Ser Val Pro His Leu Gln LysVal Phe Asp Arg Tyr Lys Ser Tyr Ser Pro Tyr Asp Met Leu GluSer Ile Arg Lys Glu Val Lys Gly Asp Leu Glu Asn Ala Phe LeuAsn Leu Val Gln Cys Ile Gln Asn Lys Pro Leu Tyr Phe Ala AspArg Leu Tyr Asp Ser Met Lys Gly Lys Gly Thr Arg Asp Lys ValLeu Ile Arg Ile Met Val Ser Arg Ser Glu Val Asp Met Leu Lys IleArg Ser Glu Phe Lys Arg Lys Tyr Gly Lys Ser Leu Tyr Tyr Tyr IleGln Gln Asp Thr Lys Gly Asp Tyr Gln Lys Ala Leu Leu Tyr LeuCys Gly Gly Asp Asp 21 CA-001Met Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu GlyAsp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Ala Tyr ThrAsn Phe Asp Ala Glu Arg Asp Ala Leu Asn Ile Glu Thr Ala IleLys Thr Lys Gly Val Asp Glu Val Thr Ile Val Asn Ile Leu Thr AsnArg Ser Asn Ala Gln Arg Gln Asp Ile Ala Phe Ala Tyr Gln ArgArg Thr Lys Lys Glu Leu Ala Ser Ala Leu Lys Ser Ala Leu SerGly His Leu Glu Thr Val Ile Leu Gly Leu Leu Lys Thr Pro AlaGln Tyr Asp Ala Ser Glu Leu Lys Ala Ser Met Lys Gly Leu GlyThr Asp Glu Asp Ser Leu Ile Glu Ile Ile Cys Ser Arg Thr Asn GlnGlu Leu Gln Glu Ile Asn Arg Val Tyr Lys Glu Met Tyr Lys ThrAsp Leu Glu Lys Asp Ile Ile Ser Asp Thr Ser Gly Asp Phe ArgLys Leu Met Val Ala Leu Ala Lys Gly Arg Arg Ala Glu Asp GlySer Val Ile Asp Tyr Glu Leu Ile Asp Gln Asp Ala Arg Asp Leu TyrAsp Ala Gly Val Lys Arg Lys Gly Thr Asp Val Pro Lys Trp Ile SerIle Met Thr Glu Arg Ser Val Pro His Leu Glu Lys Val Phe AspArg Tyr Lys Ser Tyr Ser Pro Tyr Asp Met Leu Glu Ser Ile Arg LysGlu Val Lys Gly Asp Leu Glu Asn Ala Phe Leu Asn Leu Val GlnCys Ile Gln Asn Lys Pro Leu Tyr Phe Ala Asp Arg Leu Tyr AspSer Met Lys Gly Lys Gly Thr Arg Asp Lys Val Leu Ile Arg IleMet Val Ser Arg Ser Glu Val Asp Met Leu Lys Ile Arg Ser GluPhe Lys Arg Lys Tyr Gly Lys Ser Leu Tyr Tyr Tyr Ile Gln GlnAsp Thr Lys Gly Asp Tyr Gln Lys Ala Leu Leu Tyr Leu Cys Gly Gly Asp Asp22 MS-001 Met Glu Lys Met Leu Ala Gly Cys Phe Leu Leu Ile Leu Gly GlnIle Val Leu Leu Pro Ala Glu Ala Arg Glu Arg Ser Arg Gly ArgSer Ile Ser Arg Gly Arg His Ala Arg Thr His Pro Gln Thr Ala LeuLeu Glu Ser Ser Cys Glu Asn Lys Arg Ala Asp Leu Val Phe Ile IleAsp Ser Ser Arg Ser Val Asn Thr His Asp Tyr Ala Lys Val LysGlu Phe Ile Val Asp Ile Leu Gln Phe Leu Asp Ile Gly Pro Asp ValThr Arg Val Gly Leu Leu Gln Tyr Gly Ser Thr Val Lys Asn GluPhe Ser Leu Lys Thr Phe Lys Arg Lys Ser Glu Val Glu Arg AlaVal Lys Arg Met Arg His Leu Ser Thr Gly Thr Met Thr Gly LeuAla Ile Gln Tyr Ala Leu Asn Ile Ala Phe Ser Glu Ala Glu Gly AlaArg Pro Leu Arg Glu Asn Val Pro Arg Val Ile Met Ile Val Thr AspGly Arg Pro Gln Asp Ser Val Ala Glu Val Ala Ala Lys Ala ArgAsp Thr Gly Ile Leu Ile Phe Ala Ile Gly Val Gly Gln Val Asp PheAsn Thr Leu Lys Ser Ile Gly Ser Glu Pro His Glu Asp His ValPhe Leu Val Ala Asn Phe Ser Gln Ile Glu Thr Leu Thr Ser ValPhe Gln Lys Lys Leu Cys Ile Asn Ile Met Cys Ser Thr Leu GluHis Asn Cys Ala His Phe Cys Ile Asn Ile Pro Gly Ser Tyr Val CysArg Cys Lys Gln Gly Tyr Ile Leu Asn Ser Asp Gln Thr Thr CysArg Ile Gln Asp Leu Cys Ala Met Glu Asp His Asn Cys Glu GlnLeu Cys Val Asn Val Pro Gly Ser Phe Val Cys Gln Cys Tyr SerGly Tyr Ala Leu Ala Glu Asp Gly Lys Arg Cys Val Ala Val AspTyr Cys Ala Ser Glu Asn His Gly Cys Glu His Glu Cys Val AsnAla Asp Gly Ser Tyr Leu Cys Gln Cys His Glu Gly Phe Ala LeuAsn Pro Asp Lys Lys Thr Cys Thr Lys Ile Asp Tyr Cys Ala SerSer Asn His Gly Cys Gln His Glu Cys Val Asn Thr Asp Asp SerTyr Ser Cys His Cys Leu Lys Gly Phe Thr Leu Asn Pro Asp LysLys Thr Cys Arg Arg Ile Asn Tyr Cys Ala Leu Asn Lys Pro GlyCys Glu His Glu Cys Val Asn Met Glu Glu Ser Tyr Tyr Cys ArgCys His Arg Gly Tyr Thr Leu Asp Pro Asn Gly Lys Thr Cys SerArg Val Asp His Cys Ala Gln Gln Asp His Gly Cys Glu Gln LeuCys Leu Asn Thr Glu Asp Ser Phe Val Cys Gln Cys Ser Glu GlyPhe Leu Ile Asn Glu Asp Leu Lys Tyr Cys Ser Arg Val Asp TyrCys Leu Leu Ser Asp His Gly Cys Glu Tyr Ser Cys Val Asn MetAsp Arg Ser Phe Ala Cys Gln Cys Pro Glu Gly His Val Leu ArgSer Asp Gly Lys Thr Cys Ala Lys Leu Asp Ser Cys Ala Leu GlyAsp His Gly Cys Glu His Ser Cys Val Ser Ser Glu Asp Ser PheVal Cys Gln Cys Phe Glu Gly Tyr Ile Leu Arg Glu Asp Gly LysThr Cys Arg Arg Lys Asp Val Cys Gln Ala Ile Asp His Gly CysGlu His Ile Cys Val Asn Ser Asp Asp Ser Tyr Thr Cys Glu CysLeu Glu Gly Phe Arg Leu Ala Glu Asp Gly Lys Arg Cys Arg ArgLys Asp Val Cys Lys Ser Thr His His Gly Cys Glu His Ile CysVal Asn Asn Gly Asn Ser Tyr Ile Cys Lys Cys Ser Glu Gly PheVal Leu Ala Glu Asp Gly Arg Arg Cys Lys Lys Cys Thr Glu GlyPro Ile Asp Leu Val Phe Val Ile Asp Gly Ser Lys Ser Leu Gly GluGlu Asn Phe Glu Val Val Lys Gln Phe Val Thr Gly Ile Ile Asp SerLeu Thr Ile Ser Pro Lys Ala Ala Arg Val Gly Leu Leu Gln Tyr SerThr Gln Val His Thr Glu Phe Thr Leu Arg Asn Phe Asn Ser AlaLys Asp Met Lys Lys Ala Val Ala His Met Lys Tyr Met Gly LysGly Ser Met Thr Gly Leu Ala Leu Lys His Met Phe Glu Arg SerPhe Thr Gln Gly Glu Gly Ala Arg Pro Leu Ser Thr Arg Val ProArg Ala Ala Ile Val Phe Thr Asp Gly Arg Ala Gln Asp Asp ValSer Glu Trp Ala Ser Lys Ala Lys Ala Asn Gly Ile Thr Met Tyr AlaVal Gly Val Gly Lys Ala Ile Glu Glu Glu Leu Gln Glu Ile Ala SerGlu Pro Thr Asn Lys His Leu Phe Tyr Ala Glu Asp Phe Ser ThrMet Asp Glu Ile Ser Glu Lys Leu Lys Lys Gly Ile Cys Glu AlaLeu Glu Asp Ser Asp Gly Arg Gln Asp Ser Pro Ala Gly Glu LeuPro Lys Thr Val Gln Gln Pro Thr Glu Ser Glu Pro Val Thr Ile AsnIle Gln Asp Leu Leu Ser Cys Ser Asn Phe Ala Val Gln His ArgTyr Leu Phe Glu Glu Asp Asn Leu Leu Arg Ser Thr Gln Lys LeuSer His Ser Thr Lys Pro Ser Gly Ser Pro Leu Glu Glu Lys HisAsp Gln Cys Lys Cys Glu Asn Leu Ile Met Phe Gln Asn Leu AlaAsn Glu Glu Val Arg Lys Leu Thr Gln Arg Leu Glu Glu Met ThrGln Arg Met Glu Ala Leu Glu Asn Arg Leu Arg Tyr Arg 23 MS-001Met Glu Lys Met Leu Ala Gly Cys Phe Leu Leu Ile Leu Gly GlnIle Val Leu Leu Pro Ala Glu Ala Arg Glu Arg Ser Arg Gly ArgSer Ile Ser Arg Gly Arg His Ala Arg Thr His Pro Gln Thr Ala LeuLeu Glu Ser Ser Cys Glu Asn Lys Arg Ala Asp Leu Val Phe Ile IleAsp Ser Ser Arg Ser Val Asn Thr His Asp Tyr Ala Lys Val LysGlu Phe Ile Val Asp Ile Leu Gln Phe Leu Asp Ile Gly Pro Asp ValThr Arg Val Gly Leu Leu Gln Tyr Gly Ser Thr Val Lys Asn GluPhe Ser Leu Lys Thr Phe Lys Arg Lys Ser Glu Val Glu Arg AlaVal Lys Arg Met Arg His Leu Ser Thr Gly Thr Met Thr Gly LeuAla Ile Gln Tyr Ala Leu Asn Ile Ala Phe Ser Glu Ala Glu Gly AlaArg Pro Leu Arg Glu Asn Val Pro Arg Val Ile Met Ile Val Thr AspGly Arg Pro Gln Asp Ser Val Ala Glu Val Ala Ala Lys Ala ArgAsp Thr Gly Ile Leu Ile Phe Ala Ile Gly Val Gly Gln Val Asp PheAsn Thr Leu Lys Ser Ile Gly Ser Glu Pro His Glu Asp His ValPhe Leu Val Ala Asn Phe Ser Gln Ile Glu Thr Leu Thr Ser ValPhe Gln Lys Lys Leu Cys Thr Ala His Met Cys Ser Thr Leu GluHis Asn Cys Ala His Phe Cys Ile Asn Ile Pro Gly Ser Tyr Val CysArg Cys Lys Gln Gly Tyr Ile Leu Asn Ser Asp Gln Thr Thr CysArg Ile Gln Asp Leu Cys Ala Met Glu Asp His Asn Cys Glu GlnLeu Cys Val Asn Val Pro Gly Ser Phe Val Cys Gln Cys Tyr SerGly Tyr Ala Leu Ala Glu Asp Gly Lys Arg Cys Val Ala Val AspTyr Cys Ala Ser Glu Asn His Gly Cys Glu His Glu Cys Val AsnAla Asp Gly Ser Tyr Leu Cys Gln Cys His Glu Gly Phe Ala LeuAsn Pro Asp Lys Lys Thr Cys Thr Lys Ile Asp Tyr Cys Ala SerSer Asn His Gly Cys Gln His Glu Cys Val Asn Thr Asp Asp SerTyr Ser Cys His Cys Leu Lys Gly Phe Thr Leu Asn Pro Asp LysLys Thr Cys Arg Arg Ile Asn Tyr Cys Ala Leu Asn Lys Pro GlyCys Glu His Glu Cys Val Asn Met Glu Glu Ser Tyr Tyr Cys ArgCys His Arg Gly Tyr Thr Leu Asp Pro Asn Gly Lys Thr Cys SerArg Val Asp His Cys Ala Glu Glu Asp His Gly Cys Glu Gln LeuCys Leu Asn Thr Glu Asp Ser Phe Val Cys Gln Cys Ser Glu GlyPhe Leu Ile Asn Glu Asp Leu Lys Thr Cys Ser Arg Val Asp TyrCys Leu Leu Ser Asp His Gly Cys Glu Tyr Ser Cys Val Asn MetAsp Arg Ser Phe Ala Cys Gln Cys Pro Glu Gly His Val Leu ArgSer Asp Gly Lys Thr Cys Ala Lys Leu Asp Ser Gly Ala Leu GlyAsp His Gly Cys Glu His Ser Cys Val Ser Ser Glu Asp Ser PheVal Cys Gln Cys Phe Glu Gly Tyr Ile Leu Arg Glu Asp Gly LysThr Cys Arg Arg Lys Asp Val Cys Gln Ala Ile Asp His Gly CysGlu His Ile Cys Val Asn Ser Asp Asp Ser Tyr Thr Cys Glu CysLeu Glu Gly Phe Arg Leu Ala Glu Asp Gly Lys Arg Cys Arg ArgLys Asp Val Cys Lys Ser Thr His His Gly Cys Glu His Ile CysVal Asn Asn Gly Asn Ser Tyr Ile Cys Lys Cys Ser Glu Gly PheVal Leu Ala Glu Asp Gly Arg Arg Cys Lys Lys Cys Thr Glu GlyPro Ile Asp Leu Val Phe Val Ile Asp Gly Ser Lys Ser Leu Gly GluGlu Asn Phe Glu Val Val Lys Gln Phe Val Thr Gly Ile Ile Asp SerLeu Thr Ile Ser Pro Lys Ala Ala Arg Val Gly Leu Leu Gln Tyr SerThr Gln Val His Thr Glu Phe Thr Leu Arg Asn Phe Asn Ser AlaLys Asp Met Lys Lys Ala Val Ala His Met Lys Tyr Met Gly LysGly Ser Met Thr Gly Leu Ala Leu Lys His Met Phe Glu Arg SerPhe Thr Gln Gly Glu Gly Ala Arg Pro Leu Ser Thr Arg Val ProArg Ala Ala Ile Val Phe Thr Asp Gly Arg Ala Gln Asp Asp ValSer Glu Trp Ala Ser Lys Ala Lys Ala Asn Gly Ile Thr Met Tyr AlaVal Gly Val Gly Lys Ala Ile Glu Glu Glu Leu Gln Glu Ile Ala SerGlu Pro Thr Asn Lys His Leu Phe Tyr Ala Glu Asp Phe Ser ThrMet Asp Glu Ile Ser Glu Lys Leu Lys Lys Gly Ile Cys Glu AlaLeu Glu Asp Ser Asp Gly Arg Gln Asp Ser Pro Ala Gly Glu LeuPro Lys Thr Val Gln Gln Pro Thr Val Gln His Arg Tyr Leu PheGlu Glu Asp Asn Leu Leu Arg Ser Thr Gln Lys Leu Ser His SerThr Lys Pro Ser Gly Ser Pro Leu Glu Glu Lys His Asp Gln CysLys Cys Glu Asn Leu Ile Met Phe Gln Asn Leu Ala Asn Glu GluVal Arg Lys Leu Thr Gln Arg Leu Glu Glu Met Thr Gln Arg MetGlu Ala Leu Glu Asn Arg Leu Arg Tyr Arg 24 MS-001variableGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Glyzone in heavyGlu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr chainAsn Tyr Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly LeuGlu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr SerPro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile SerThr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr AlaMet Tyr Tyr Cys Ala Arg Asp Tyr Trp Tyr Lys Pro Leu Phe AspIle Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 25 MS-001variableAsp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leuzone in lightGly Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr chainSer Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys ProGly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu SerGly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr TyrCys Gln Gln Tyr Ser Ser Phe Pro Ile Tyr Phe Gly Gln Gly ThrLys Val Glu Ile Lys Arg 26 MS-001variableGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Glyzone in heavyGly Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser chainSer Tyr Gly Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Ile Gly Trp Ile Asp Pro Gly Ser Gly Gly Thr Lys Tyr AsnGlu Lys Phe Lys Gly Lys Ala Thr Ile Ser Arg Asp Asn Ser LysAsn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys Ala Arg Glu Arg Tyr Gly Tyr Tyr Phe AspTyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 27MS-001variableGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Glyzone in heavyGly Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser chainSer Tyr Gly Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Ile Gly Trp Ile Asp Pro Gly Ser Gly Gly Thr Lys Tyr AsnGlu Lys Phe Lys Gly Lys Ala Thr Ile Ile Ser Arg Asp Asn Ser LysAsn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys Ala Arg Glu Arg Tyr Gly Tyr Tyr Phe AspTyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 28 MS-001variableGlu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Glyzone in lightGlu Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Tyr chainLeu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuIle Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Val Trp Asp SerSer Pro Pro Val Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 29MS-001variableGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Glyzone in heavyGly Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr chainSer Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Ile Gly Arg Ile Asn Pro Asp Ser Gly Ser Thr Lys Tyr AsnGlu Lys Phe Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser LysAsn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys Ala Arg Gly Gly Arg Leu Ser Trp Asp PheAsp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 30 MS-001variableAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Glyzone in lightAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg Tyr chainLeu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe SerGly Ser Gly Ser Gly thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Ala Tyr Asp TyrSer Leu Gly Gly Tyr Val Phe Gly Asp Gly Thr Lys Val Glu Ile Lys 31MS-001variableAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Glyzone in lightAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg Tyr chainLeu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Ala Tyr Asp TyrSer Leu Gly Gly Tyr Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 32MS-001variableGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Glyzone in heavySer Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Ser chainHis Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu TrpMet Gly Gly Ile Asn Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln LysPhe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr AlaTyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val TyrTyr Cys Ala Arg His Tyr Glu Ile Gln Ile Gly Arg Tyr Gly Met AsnVal Tyr Tyr Leu Met Tyr Arg Phe Ala Ser Trp Gly Gln Gly ThrLeu Val Thr Val Ser Ser 33 MS-001variableAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Glyzone in lightAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Ser Ala chainLeu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr Asn Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp GlyAsp Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 34 LY3015014Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Heavy chainGly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe SerLys Leu Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Val Ser Thr Ile Ser Ser Gly Gly Gly Tyr Thr Tyr Tyr ProAsp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala LysAsn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys Ala Arg Glu Gly Ile Ser Phe Gln Gly Gly ThrTyr Thr Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala ProCys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys LeuVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser AsnThr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro CysPro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg ThrPro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp ProGlu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His AsnAla Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr ThrPro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser ArgLeu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe SerCys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln LysSer Leu Ser Leu Ser Leu Gly 35 LY3015014 LightAsp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly chainGlu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His ArgAsn Gly Ile Thr Tyr Ser Tyr Trp Tyr Leu Gln Lys Pro Gly Gln SerPro Gln Leu Leu Ile Tyr Gln Leu Ser Asn Leu Ala Ser Gly ValPro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuLys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys TyrGln Asn Leu Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys ValGlu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro ProSer Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys LeuLeu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys ValAsp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr GluGln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu ThrLeu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys GluVal Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe AsnArg Gly Glu Cys 36 NS-001 variableGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Glyzone in heavyAla Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Thr chainMet Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu GluTrp Met Gly Arg Ile Asp Pro Ala Asn Glu His Thr Asn Tyr AlaGln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser IleSer Thr Ala Tyr Met Glu Leu Ser Arg Leu Thr Ser Asp Asp ThrAla Val Tyr Tyr Cys Ala Arg Ser Tyr Tyr Tyr Tyr Asn Met AspTyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 37 NS-001 variableGln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Glyzone in lightGlu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Tyr chainMet His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuIle Tyr Gly Val Phe Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Gly Arg LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Trp Ser SerAsp Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 38NS-001 variableGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Glyzone in  heavyAla Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Thr chainMet Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu GluTrp Met Gly Arg Ile Asp Pro Ala Asn Glu His Thr Asn Tyr AlaGln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser IleSer Thr Ala Tyr Met Glu Leu Ser Arg Leu Thr Ser Asp Asp ThrAla Val Tyr Tyr Cys Ala Arg Ser Tyr Tyr Tyr Tyr Asn Met AspTyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 39 NS-001 variableGlu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Glyzone in lightGlu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Tyr chainMet His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuIle Tyr Gly Val Phe Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Gly Arg LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Trp Ser SerAsp Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 40NS-001 variableGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Glyzone in heavyAla Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Thr chainMet Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu GluTrp Met Gly Arg Ile Asp Pro Ala Asn Glu His Thr Asn Tyr AlaGln Lys Phe Gln Gly Arg Val Thr Met Thr Asg Asp Thr Ser IleSer Thr Ala Tyr Met Glu Leu Ser Arg Leu Thr Ser Asp Asp ThrAla Val Tyr Tyr Cys Ala Arg Ser Tyr Tyr Tyr Tyr Ala Met Asp TyrTrp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 41 NS-001 variableGln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Glyzone in lightGlu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Tyr chainMet His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuIle Tyr Gly Val Phe Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Gly Arg LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Trp Ser Ser Asp Pro Pro

Beneficial Effect

The invention provides a new and better treatment method for thetreatment of inflammatory immune diseases. Through the disclosure of theinvention, the system or topical PCSK9 inhibitor can be furtherprepared, and a new monomer drug or compound preparation containingvarious PCSK9 inhibitors can be developed. Furthermore, new monomers orcompound formulations containing all kinds of PCSK9 inhibitors have beendeveloped for the treatment of various types of inflammatory immunediseases, especially psoriasis, eczema and urticaria. Clinical trialshave shown that these drugs, which contain PCSK9 inhibitors, areeffective, have little adverse reactions, and are well tolerated inpatients. In particular, topical use alone can significantly improvelesions in immune diseases such as psoriasis. It is very suitable forthe current clinical demand and is expected to have great applicationprospect, which will bring more advantages to the patients withinflammatory and immunological diseases.

V. EXAMPLES

The following specific examples illustrate the embodiments of thisinvention. Technical personnel in this field can easily understand theother advantages and effects of the invention from the contentsdisclosed in this invention. The invention is not limited to theembodiments described herein. Before further describing the embodimentsof this invention, it should be understood that the scope of protectionof the invention is not limited to the following specific embodiment. Itshould be also understood that the term used in the specific examples ofthe invention is used to describe a specific embodiment rather than tolimit the protection scope of the invention. In the description andclaims of the invention, unless expressly stated in the text, thesingular form “one” and “this” include plural forms.

It should be understood that when a numerical range is given in anexample, unless stated by the invention, the two endpoints and anybetween the two endpoints of each numerical range can be selected.Unless defined herein, all technical and scientific terms used in theinvention have the same meaning as those commonly understood by those inthe technical field. Except for specific methods, equipment, materialsused in the embodiment, according to the description of the invention,one skilled in the art would understand the use of the equivalents ofthe methods, equipment, and materials described herein.

Example 1 PCSK9 Knockout Significantly Alleviates Psoriasis-LikeInflammation Reaction Induced by IMQ in Mice

Reagents: 5% imiquimod cream (IMQ), PCSK9 antibody (abcam), NF-kBantibody(abcam)

Experimental animal: C57BL/6 (B6) mice, 7 male; C57BL/6-PCSK9-r-mice, 5males and 5 females. The mice were purchased at the Jackson Institute inMaine, USA (The Jackson Laboratory).

Experimental Methods:

1. After two groups of mice with different genotypes were treated withback hair removal, 5% Imiquimod cream of 62.5 mg was applied daily for 5days.

2. Scores (the score of erythema, scales, skin lesions thickening andtotal score) were taken and archived (the score was obtained by tworesearchers respectively then average score was calculated) before andafter the application.

3. On the last day of the experiment, all the mice were eulogized andthe skin tissue of the back (treated and untreated) was taken.

4. The morphology of skin lesions in each group was observed by HEstaining and the expression and distribution of PCSK9 and NF-kB in skinlesions of 4 groups were measured by immunofluorescence method.

Experimental Results:

1. After 5 days of continuous application, there were obvious erythema,scales and infiltration in the back of 7 C57BL/6 mice, which were inaccordance with the appearance of psoriasis-like skin lesions. In 10PCSK9 knockout mice, there were only slight erythema, scales andinfiltration in the back coating area (See FIG. 1).

2. A daily score was given on the skin lesions of the drug-coated areaon the back of the mice, the score of erythema, scale, infiltration andtotal score of C57BL/6 mice were significantly higher than those ofPCSK9 knockout mice (P<0.05).

3. After 5 days of application, the skin tissues of the mice's back(treated and untreated) were taken for HE staining and pathologicalchanges were observed. The skin of IMQ treatment area of C57BL/6 miceshowed typical psoriasis pathological changes, such as skin thickening,dermatoid lengthening, thickening of spinous layer, hyperkeratosis withincomplete keratosis, Kogoj abscess and Munro abscess, etc. However, theskin of PCSK9 gene knockout mice showed mild epidermis thickening andhyperkeratosis, no typical pathological changes of psoriasis, such asextension of dermatoid, thickening of spinous layer, hyperkeratosis withincomplete keratosis, Kogoj abscess and Munro abscess. The untreatedskin of both groups showed normal skin structure (see FIG. 3).

Example 2 Transfection of Si-PCSK9 Enhances Apoptosis and InhibitsProliferation of Human Keratinocytes.

Experimental Materials and Reagents:

(1) Human primary keratinocytes (Lifeline Cell Technology, FC-0064)

(2) DermaLife Culture medium of keratinocyte (Lifeline Cell Technology,LL-0007), si-PCSK9 (Santa Cruz,sc-45482), Lipofectamine 3000transfection reagent (ThermalFisher, L-3000001), AnnexinV (BD), PI(BD).

Experimental Methods:

1. Cultured human keratinocytes were replanted in 6 wells plates. Whenthe cell density was 60-70%, si-RNA (si-Con and siPCSK9) transfectionwas carried out. After 24 h/48 h/72 h of transfection, measured the cellactivity of each pore by MTT method. 3 wells were selected at each timepoint for each group, the average value was taken and the cell activitycurve was plotted.

2. Cultured human keratinocytes were replanted in 6 wells plates. Whenthe cell density was 60-70%, si-RNA (si-Con & si-PCSK9) transfection wascarried out. After 24 h/48 h/72 h of transfection, cells were harvestedrespectively for AnnexinV and PI staining to detect cell apoptosis andcell cycle. 3 wells were selected at each time point for each group, theaverage value was taken.

Experimental Results:

1. The number of survival cells of human primary keratinocytestransfected with si-PCSK9 was significantly lower than that of si-Contransfection (P<0.05, See FIG. 6).

2. The apoptosis of primary keratinocytes transfected with si-PCSK9 wassignificantly higher than that of si-Con transfection (P<0.05), whileproliferating (the proportion of S+G2/M phase cells) decreasedsignificantly (see FIG. 7).

Example 3

The Expression of PCSK9 in Psoriatic Lesions was Significantly Higherthan that in Non-Lesions and Normal Controls.

Experiment materials: lesions and non-lesion from 30 psoriasis patients,skin samples from 30 normal people were treated with PCSK9 antibody(abcam).

Methods: immunohistochemistry, real time quantitative-PCR.

Results: the skin of patients with skin lesions and non-lesions wasextracted by drilling method. The skin of normal people originated fromthe redundant skin of cosmetic surgery. The results ofimmunohistochemistry showed that the expression of PCSK9 in psoriaticlesions was significantly higher than that in non-lesions and normalcontrols. PCSK9 positive cells were mainly distributed in epidermis anddermis near epidermis, but not in dermis vessels (see FIG. 8). RNA wasextracted from skin homogenate. Q-PCR detection showed that theexpression of PCSK9 in psoriatic lesions group was higher than that inpsoriatic non-lesion group and normal control group (P<0.05) (see FIG.9). The results of immunohistochemistry and Q-PCR were consistent.

Example 4

Expression of PCSK9 in Peripheral Blood Mononuclear Cells and Plasma inPatients with Psoriasis, Eczema and Urticaria.

Materials: 5 ml peripheral blood from 30 cases of psoriasis, 30 cases ofeczema, 30 cases of urticaria, and 30 cases of normal persons wascollected respectively.

Empirical Method:

(1) Separation of plasma and separation of peripheral PBMC by densitygradient method.(2) Detection of PCSK9 expression in PBMC by real-time quantitativeQ-PCR.(3) The expression of PCSK9 in plasma was detected by enzyme linkedimmunosorbent assay (Elisa) (PCSK9 Quantikine ELISA Kit, America R&D)(4) Results: (1) No PCSK expression was detected in PBMC of the samplesof psoriasis, eczema, urticarial and normal people.(5) PCSK expression in the samples of psoriasis, eczema, and urticariaare much higher than that in the sample of the normal people. PCSKexpression is the highest in the samples of psoriasis.

Example 5

Effect of PCSK9 Protein on the Secretion of IL-17 and IL-2 fromPeripheral CD4+ T Cells in Patients with Psoriasis, Eczema or Urticaria.

Materials: peripheral blood of 10 cases of psoriasis, 10 cases ofeczema, 10 cases of urticaria, and 10 cases of normal people. ELISA kitwas purchased from Raybiotech Company of USA.

Experimental Methods:

(1) Isolation of CD4⁺T cells from peripheral blood:

After PBMC was isolated by Ficoll-Hypaque from human peripheral blood bydensity gradient centrifugation, washed with 10 times volume 1×BD beadsbuffer. Then 50 ul BD IMag TM CD4 beads were added to each 107 cells.The beads were mixed well and incubated at room temperature for 30minutes. 1 ml 1×BD magnetic bead buffer solution was added, and thecells were transferred to the round bottom tube and placed in themagnetic frame for 8 to 10 minutes. And then the supernatant wasdiscarded and removed the test tube from the magnetic field. Afterresuspension of cells attached to tube wall with 1 ml 1×BD magnetic beadbuffer, the tubes were placed in magnetic field for 2-4 min again. Thesupernatant was discarded removed it out of the magnetic field,resuspended the cells and placed the tube in the magnetic field for 2-4min. The cells obtained from the supernatant can be used in subsequentexperiments. BD IMag TM CD4 separation system was purchased from BDBiosciences Company.

(2) Determination of Cytokines Secreted by CD4⁺T cells in PeripheralBlood

The supernatant was collected after cell culture under differentconditions. IFN-Gamma represented TH1 type cytokines, IL-4 representedTH2 type cytokines, and IL-17 represented TH17 cytokines. The detectionof IFN-Gamma, IL-4, IL-17 was conducted by ELISA.

Results: Compared to the normal patients, PCSK9 protein cansignificantly promote the secretion of IL-17, IL-2 and IFN-Gamma(P<0.01) by CD4 T cells isolated from peripheral blood of patients withpsoriasis, eczema and urticaria (FIG. 11).

Example 6

Subcutaneous Injection of PCSK9 Small Molecule Inhibitor and PCSK9Monoclonal Antibody Significantly Alleviated Systemic ImmuneAbnormalities and Skin Lesions Induced by IMQ in Mice. The Efficacy ofPCSK9 Small Molecule Inhibitor is Better than that of SubcutaneousInjection of PCSK9 Monoclonal Antibody.

Reagents:

The PCSK9 small molecule inhibitor used for this example has the formulaof:

The PCSK9 monoclonal antibody used for this example is Evolocumab. 40SPF female BALB/c mice aged 6-8 weeks were randomly divided into normalcontrol group, model group, PCSK9 small molecule inhibitor group andPCSK9 monoclonal antibody group. There were 10 mice for each group.After intraperitoneal injection of pentobarbital sodium 80 mg/kg, theback was shaved with an area of about 2 cm×3 cm. The mice were feedseparately for 1 day.

(1) In normal control group, Vaseline was applied locally. In modelgroup, inhibitor group and monoclonal antibody group, 62.5 mg of 5%imiquimod cream was applied on the back every day for 6 consecutivedays, photos were taken and the PASI score was evaluated.

(2) On the first day, normal saline was subcutaneously administrated tonormal control group and model group, PCSK9 small molecule inhibitor wassubcutaneously administrated (8 mg/kg, Purchased from Selleck Inc.) toinhibitor group, and PCSK9 monoclonal antibody was subcutaneouslyadministrated to monoclonal antibody group (10 mg/kg, Purchased fromabcam Inc.)

Experimental Results:

(1) After 6 days of continuous application, there were marked erythema,scales and infiltration in the back of the mice in the model group.However, in PCSK9 small molecule inhibitor group and PCSK9 monoclonalantibody group, there was only mild erythema, scales and infiltration inthe back coating area of mice. The effect of PCSK9 small moleculeinhibitor group was better than that of PCSK9 monoclonal antibody group.No lesions were observed in the vaseline group (FIG. 12).

(2) A daily scoring was given on the skin lesions of the drug-coatedarea on the back of the mice. The score of erythema, scales,infiltration and total score of PCSK9 small molecule inhibitor group(erythema+scales+infiltration) were significantly lower than that ofPCSK9 monoclonal antibody group (P<0.01).

Example 7

Both PCSK9 Small Molecule Inhibitor and PCSK9 Monoclonal AntibodyImproved the IMQ Induced Psoriatic Lesions of Mice, and the Efficacy ofSmall Molecule Inhibitor was Better than that of Monoclonal Antibody

Reagents:

The PCSK9 small molecule inhibitor used for this example has the formulaof:

The PCSK9 monoclonal antibody used for this example is Evolocumab.

(1) 40 SPF female BALB/c mice aged 6-8 weeks were randomly divided intonormal control group, model group, PCSK9 inhibitor group and PCSK9monoclonal antibody group. There were 10 mice for each group. Afterintraperitoneal injection of pentobarbital sodium 80 mg/kg, the back wasshaved with an area of about 2 cm×3 cm. The mice were feed separatelyfor 1 day.

(2) In normal control group, vaseline was applied locally. In modelgroup, small molecule inhibitor group and monoclonal antibody group,62.5 mg of 5% imiquimod cream was applied on the back every day for 6consecutive days, and photos were taken and the PASI score wasevaluated.

(3) Pretreatment: vaseline was applied on the back skin with mics innormal control group and model group, PCSK9 small molecule inhibitorcream (0.01% concentration) was applied to inhibitor group once daily.And, PCSK9 monoclonal antibody cream (0.01% concentration) was appliedto monoclonal antibody group once daily.

(4) After 7 days pretreatment, vaseline was applied on the back skinwith mice in normal control group. At the same time, 62.5 mg 5%imiquimod cream was regularly applied daily on the back with mice inmodel group, small molecule inhibitor group and monoclonal antibodygroup. PCSK9 small molecule inhibitor cream or PCSK9 monoclonal antibodycream was applied after one hour. For 6 consecutive days, photos weretaken and the PASI score was evaluated.

Experimental Results:

1. After 6 days of continuous administration, erythema, scales andinfiltration were observed in the coating areas at the back of the micsin the model group. However, there were only slight erythema, scales andinfiltration in the back of the mice in the PCSK9 small moleculeinhibitor group and the PCSK9 monoclonal antibody group. The effect ofPCSK9 small molecule inhibitor group was better than that of PCSK9monoclonal antibody group. No lesion was observed in the vaseline group(See FIG. 13).

2. A daily score was given on the skin lesions of the drug-coated areaon the back of the mice. The score of erythema, scales, infiltration andtotal score (erythema+scales+infiltration) in PCSK9 small moleculeinhibitor group was significantly lower than that in PCSK9 monoclonalantibody group (P<0.01).

Example 8

External Use of siRNA for PCSK9 could Significantly Reduce PsoriaticLesions Induced by IMQ in Mice.

Materials and Methods:

1. Animal models: two types of mice models, C57BL/6J (B6) and Balb/cByJ(BALB) were used in this study. The mice was purchased at the JacksonInstitute in Maine, USA. (The Jackson Laboratory). The strain number was000664, 001026 and 005993 respectively (www.jax.org). All the mice inthis experiment were female, aged 3-8 months, 5 mice for each group.

2. Psoriatic lesions induced by IMQ: the back hair of B 6 mice wasremoved one day before the experiment. During the experiment, 62.5 mgimiquimod (IMQ, 3 M pharmaceuticals) were smeared on the skin of eachmouse with an area of 2 cm² on the back. In some mice, right auricle wasalso smeared with 5 mg IMQ to detect skin thickening easily. After 24hours, score of the skin color, thickness and scales degree wasevaluated.

3. Inhibition of PCSK9 by siRNA and its effect on psoriatic lesionsinduced by IMQ: Two siRNAs for PCSK9 (siPCSK9-1, 2) and a randomsequence siRNA (siCon), used as a control trial, was synthesized bySigma-Aldrich company (Sigma-Aldrich, USA). The sequence was shown inTable 3. Mixed siPCSK9-1 and 2 equally and diluted it to 20 μM withnormal saline. 12.5 μl diluted siPCSK9 was evenly mixed with 7.5 μlmoisturizer (CVS Pharmacy, Baby Lotion), so it was done with siCon.Applied 20 μl siRNA-moisturizer mixture to the skin 1 hour before IMQwas applied. PCSK9 monoclonal antibody was purchased from abcam Companyand was evenly mixed with PCSK9 moisturizer (CVS pharmacy, Baby Lotion)to generate an equal concentration emulsion with siPCSK9 emulsion.

4. From the first day, siCON was applied on the back skin of B6 mice inmodel group after 1 hour-vaseline treatment. B6 mice in siPCSK9 groupwere smeared with 5% imiquimod cream on their back, then 1 hourthereafter, siPCSK9 was applied once daily. In PCSK9 monoclonal antibodygroup, 5% imiquimod cream was applied on the back skin of B6 mice, then1 hour thereafter, PCSK9 monoclonal antibody was applied once daily.

5. Gene expression detection by semi quantitative (real-time PCR):Primer sequence was shown in Table 3

TABLE 3 siRNA sequence and modification Gene 5′-3′ Sense 5′-3′ AntisensesiPCSK9-1 GccuGGAGuuuAuu UUCCgAAuAAACUC cGGAAdT*dT cAGGCdT*dT(SEQ ID NO: 42) (SEQ ID NO: 45) siPCSK9-2 AGGuGuAucuccuA GUGUCuAGGAGAuAGAcAcdT*dT cACCUdT*dT (SEQ ID NO: 43) (SEQ ID NO: 46) siCONcuuAcGcuGAGuAc UCGAAGuACUcAGC uucGAdT*dT GuAAGdT*dT (SEQ ID NO: 44)(SEQ ID NO: 47) Note: lowercase letters denote 2′-OMe modification. Allsequences were connected with phosphorothioate at the end of thesequence.

TABLE 4 Primer sequence Gene Forward Sequence Reverse Sequence PCSK9TTGCAGCAGCTGGGAACTT CCGACTGTGATGACCTCTGGA (SEQ ID NO: 48)(SEQ ID NO: 53) NFkB CTGGTGGACACATACAGGAA ATAGGCACTGTCTTCTTTCACGAC (SEQ ID NO: 49) CTC (SEQ ID NO: 54) IL-17A TTTTCAGCAAGGAATGTGGATTCATTGTGGAGGGCAGAC (SEQ ID NO: 50) (SEQ ID NO: 55) IL-22TTTCCTGACCAAACTCAGCA CTGGATGTTCTGGTCGTCAC (SEQ ID NO: 51)(SEQ ID NO: 56) IL-23 CACCTCCCTACTAGGACTCA TGGGCATCTGTTGGGTCTGC (SEQ ID NO: 52) (SEQ ID NO: 57)

Experimental Results:

1. siPCSK9 can significantly reduce the expression of PCSK9 in the skinof B6 mice one day after being applied to the skin (p<0.05); inhibitionrate is over 80% (FIG. 14). A day after siPCSK9 treatment of skin, itcan inhibit the expression of NF K B, IL-17, IL-22, IL-23 and otherimmune factors (Figure. 15). The expression of these inflammatoryfactors is closely related to the pathogenesis and severity ofpsoriasis, suggesting that siPCSK9 can inhibit the occurrence ofpsoriasis and alleviate the pathological reaction.

2. After 6 days of continuous application, there were significanterythema, scales and infiltration in the coating area of the modelgroup, but only slight erythema, scale and infiltration in the siPCSK9group (see FIG. 16). The therapeutic effect of siPCSK9 group onpsoriasis lesions in mice was significantly better than that in controlgroup (P<0.01), but no lesions were found in Vaseline group.

3. Daily scoring of skin lesions in B6 mice showed that the scores oferythema, scale, infiltration and the total scores(erythema+scale+infiltration) in siPCSK9 group were significantly lowerthan those in control group (P<0.01) (FIG. 16).

4. To further verify the inhibitory effect of siPCSK9 on psoriaticlesions induced by IMQ, we repeated the experiment in FIG. 16 in anotherwild mice BALB using the same treatment, and the results was verysimilar (FIG. 17).

The specific embodiments are described above in detail. Within theknowledge of the technical personnel in this field, various changes canalso be made without departing from the concept of the invention.

1. A method of treating an inflammatory immune disease by administrationof a therapeutically effective amount of a proprotein convertasesubtilisin kexin 9 (PCSK9) inhibitor selected from the group consistingof PCSK9 monoclonal antibodies, PCSK9 small molecule interfering RNAs,and a PCSK9 vaccine, and the inflammatory immune disease is selectedfrom the group consisting of psoriasis, psoriatic arthritis, eczema,atopic dermatitis, urticaria, glucocorticoid dependent dermatitis,rheumatoid arthritis, scleroderma, diabetes, chronic liver disease, andlymphoma.
 2. The method of claim 1, wherein the PCSK9 inhibitor is oneor more of the PCSK9 monoclonal antibodies.
 3. The method of claim 2,wherein the inflammatory immune disease is psoriasis, atopic dermatitisor urticaria, and the PCSK9 inhibitor is topically, systemically orsubcutaneously administered.
 4. The method of claim 3, wherein theinflammatory immune disease is psoriasis.
 5. The method of claim 3,wherein the PCSK9 inhibitor is administered once per day.
 6. The methodof claim 2, wherein the PCSK9 inhibitor is topically administered. 7.The method of claim 5, wherein the PCSK9 monoclonal antibodies areadministered as an ointment in a concentration of 0.001-0.05%.
 8. Themethod of claim 1, wherein the PCSK9 inhibitor is one or more of thesmall molecule interference RNAs.
 9. The method of claim 8, wherein theone or more small molecule interference RNAs significantly inhibitabnormal proliferation of human keratinocytes and promote theirapoptosis through an NFkb pathway.
 10. The method of claim 8, whereinthe inflammatory immune disease is selected from the group consisting ofpsoriasis, psoriatic arthritis, eczema, atopic dermatitis, urticaria,glucocorticoid dependent dermatitis, and scleroderma.
 11. The method ofclaim 10, wherein the therapeutically effective amount of the PCSK9inhibitor is administered topically.
 12. The method of claim 11, whereinthe PCSK9 inhibitor is administered as a cream or ointment containingthe PCSK9 inhibitor in a concentration of 0.001-0.05%.
 13. The method ofclaim 11, wherein the PCSK9 inhibitor is administered once per day. 14.The method of claim 8, wherein the therapeutically effective amount ofthe PCSK9 inhibitor is administered subcutaneously.
 15. The method ofclaim 14, wherein the PCSK9 inhibitor is administered at a concentrationof 8 mg/kg or in a dose of 5 mg.
 16. The method of claim 8, wherein thePCSK9 inhibitor is administered subcutaneously, topically orsystemically.
 17. The method of claim 16, wherein the PCSK9 inhibitor isadministered (i) topically as a cream or ointment containing the PCSK9inhibitor in a concentration of 0.001-0.05% or (ii) subcutaneously at aconcentration of 8 mg/kg or in a dose of 5 mg.
 18. The method of claim1, wherein the PCSK9 inhibitor is a PCSK9 vaccine.
 19. The method ofclaim 18, wherein the therapeutically effective amount of the PCSK9inhibitor is administered subcutaneously.